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Wheat, Fusarium toxins and disease: the good, the bad and the ugly H. Y. (Michelle) Seow1, X. W. Guo2, W. G. Dilantha Fernando2 1 Department of Microbiology; 2 Department of Plant Science, University of Manitoba, Winnipeg, Manitoba Impact Statement: Statement: The more potent 3ADON toxintoxin-producing fusarium isolates are on the increase in Manitoba INTRODUCTION ¾THE GOOD: Wheat is the most economically important crop in Canada, with Manitoba exporting wheat to approximately 66 different countries. It is processed into flour, cereal food, animal feed and industrial products such as ethanol. ¾THE BAD: Fusarium head blight (FHB) is the most serious disease in wheat around the world. Fusarium graminearum is the causal agent of FHB which lowers wheat yield, damages grain quality, and causes animal feed refusal and illness in humans by the mycotoxins it produces- deoxynivalenol (DON), nivalenol (NIV), 3-acetyl deoxynivalenol (3 ADON) and 15-actyl deoxynivalenol (15 ADON). ¾THE UGLY: Detection of less potent toxin 15 ADON has been higher than the other toxins in Manitoba, but this might be changing with the more potent 3 ADON increasing and posing a danger to the industry. OBJECTIVES ¾Obtain evidence that pathogen toxin profiles have changed from less potent 15 ADON to more potent 3 ADON. ¾ Determine toxin profile of Fusarium graminearum strains in Manitoba wheat fields. RESULTS MATERIALS AND METHODS ¾Wheat spikes of Superb and AC Barrie varieties were collected from 31 fields in 2004 and 2005. These were plated onto potato-dextrose agar (PDA) for 5 days. ¾Mycelia which had Fusarium graminearum characteristics (red, pinkish, or orange) were transferred onto synthetic nutrient agar for spore production and single spored using water agar medium. ¾DNA extracted from mycelium of single spore culture. Primer name 3CON 3NA 3D15A 3D3A Sequence (5'-3') TGGCAAAGACTGGTTCAC GTGCACAGAATATACGAGC ACTGACCCAAGCTGCCATC CGCATTGGCTAACACATG Fg16F Fg16R Sequence (5’-3’) C D E F G H I ← ~610 bp M 1 2 3 ← ~243 bp Figure 2.Multiplex PCR products with TRI3 primers on 2% agarose gel. Lanes. M- 1kb ladder. A, H, I- Fusarium graminearum isolates that produce 3 ADON. B,C, D, E, F, G- Fusarium graminearum isolates that produce 15 ADON. CONCLUSIONS RESULTS 4 Figure 1. Amplified Fusarium graminearum specific PCR products on 2% agarose gel. Lanes. M- 1kb ladder. 1,3- Isolates that were not Fusarium graminearum. 2,4- Isolates that were Fusarium graminearum. ¾Results of multiplex PCR show that for all farms except Hamiota, the percentage of Fusarium graminearum strains that contain the gene which produces 15 ADON decreases from 2004. Inversely, isolates which contain the gene that produces 3 ADON increases from 2004 to 2005. Results of multiplex PCR for 2004 and 2005 Fusarium graminearum isolates from same location. 2005 Total 15 ADON (%) Portage la Prairie 55.88 44.44 44.12 55.56 Size (bp) Plumas Hamiota 85.71 75 60 100 14.29 25 40 0 450 Rapid City Virden 80 100 71.43 75 20 0 28.57 25 CTCCGGATATGTTGCGTCAA GGTAGGTATCCGACATGGCAA Size (bp) 840 610 243 2004 Total 15 ADON (%) ¾Fusarium graminearum were detected using primer pairs Fg16F and Fg16R specific to the species. Primer name Specificity common to all NIV specific 15ADON specific 3ADON specific ¾ For the 2004 samples, 131 isolates were confirmed to be Fusarium graminearum through PCR where they had bands around 450bp (Figure 1). Similarly, 121 isolates were confirmed to be F. graminearum from the 2005 isolates. Location PCR Analysis- Species Detection A B TRI3 specific primers ←~450 bp MATERIALS AND METHODS Grain Samples, Isolation of Single Spores and DNA Extraction M PCR Analysis- Mycotoxins ¾TRI3 specific primers were used in multiplex PCRs to detect Nivalenol, 3 ADON and 15 ADON genes. 2004 Total 3 2005 Total 3 ADON (%) ADON (%) ¾Fusarium graminearum strains in Manitoba which are able to produce 3 ADON toxins are increasing, while those which are able to produce 15 ADON strains are decreasing. ¾More studies should be done on this shift of 15 ADON to 3 ADON production, as well as on the significance and impact of this shift on the wheat industry to protect the future of the food and feed industry. ACKNOWLEDGMENTS ¾ The Natural Sciences and Engineering Research Council of Canada and Canadian Wheat Board for the funding of this project. ¾ Daniel Brick for assistance in fieldwork. ¾ Todd Ward (Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Preoria, IL) for TRI3 primer sequences.